Method for prognosing response to cancer therapy with 5, 10, 15, 20-tetrakis (carboxyphenyl) porphine

ABSTRACT

Presented is a method of prognosing a patient&#39;s response to a cancer therapy wherein prior to the therapy contacting a sample of cells from the patient&#39;s tissue or organ being treated for the cancer with a solution of TCPP to permit binding of the TCPP to components of the abnormal dysplastic or carcinomic cells, if any are present; detecting TCPP fluorescence in the sample, the presence of TCPP fluorescence being indicative that the sample contains dysplastic or carcinomic cells; at intervals during the therapy and subsequent to the therapy performing steps a-c on another sample of cells from the patient&#39;s tissue or organ being treated for the cancer; and determining if the percentage of abnormal pre-cancerous cells in the samples tested during and subsequent to the therapy are reduced as compared with the sample tested prior to the therapy, the reduction being prognostic of the patients response to the cancer therapy.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 12/133,855 entitled Composition and Method forMaking 5,10,15,20-Tetrakis (Carboxyphenyl) Porphine filed Jun. 5, 2008and issued as U.S. Pat. No. 7,670,799 on Mar. 2, 2010, which is adivisional application of U.S. patent application Ser. No. 10/933,140entitled Method for Prognosing Response to Cancer Therapy with5,10,15,20-Tetrakis (Carboxyphenyl) Porphine filed Sep. 2, 2004 andissued as U.S. Pat. No. 7,384,764 on Jun. 10, 2008, which is adivisional application of U.S. patent application Ser. No. 09/989,092entitled Compositions and Methods for Detecting Pre-Cancerous Conditionsin Cell and Tissue Samples Using 5,10,15,20-Tetrakis (Carboxyphenyl)Porphine filed Nov. 19, 2001 and issued as 6,838,248 on Jun. 4, 2005,which claims the benefit of U.S. Provisional Patent Application Ser. No.60/249,505 entitled Method of Detecting Pre-Cancerous Conditions inHuman Tissue Samples Using 5,10,15,20-Tetrakis (Carboxyphenyl) Porphine,filed on Nov. 17, 2000, and the specification thereof of each isincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the use of certain porphyrins to detectdysplastic, pre-cancerous, and cancerous cells from various tissuesamples both in vitro and in situ.

BACKGROUND OF THE INVENTION

Various scientific and scholarly articles are referred to in parenthesesthroughout the specification. These articles are incorporated byreference herein to describe the state of the art to which thisinvention pertains.

Pathologists, who examine disease progression and analyze tissue samplesfor abnormalities, including cancer, have determined that a cellularcondition called dysplasia, which refers to abnormal formation ormaturation of cells, can potentially identify cells in a pre-cancerouscondition. Unchecked, dysplasia can progress through mild, moderate andsevere stages and eventually to cancer. About one in seven of themoderate cases of dysplasia will progress to cancer, and as many as 83%of cases with severe dysplasia have been reported to progress to cancer,depending on the types of cells involved. However, removal of mild andmoderate dysplasias greatly reduces the development of cancer. In thelung, removal of dysplastic cells not only greatly reduces the formationof cancerous cells, but in some cases pulmonary tissue will return to anormal morphology.

In general, the earlier cancers are detected, the better the prognosisis for patient survival. If breast cancer is detected early when it isstill localized to a single mass, the five-year survival rate is morethan 96%. When it has spread to a distant location, the five-yearsurvival rate is less than 20%. For lung cancer, when it is detected asa single mass the 5-year survival is more than 46%. When it has spread,the five-year survival is less than 14%. For cervical cancer, additionalimprovement in survival occurs when pre-cancerous changes are found andtreated before developing into a more severe stage (Boring and Squires1993, CA Cancer J Clin 43:7-26 and Ferguson 1990, Hematol Oncol Clin NAm 4:1053-1168).

Lung carcinoma is presently the leading cause of cancer mortality amongmen and women in the United States (Wingo et al. 1995, CA Clinical JClin 45.8-30). In 1997, there were an estimated 160,000 deaths from lungcancer, accounting for 12% of all cancer deaths in U.S. men and 2% inU.S. women (Boring & Squires 1993, supra). Lung cancer is also one ofthe most lethal types of cancer, as reflected in a five-year survivalrate of only 14%. The poor prognosis for lung cancer patients, relativeto other types of human cancer, is due largely to the lack of effectiveearly detection methods. At the time of clinical (symptomatic)presentation, over two thirds of all patients have regional noduleinvolvement or distant metastases, both of which are usually incurable.In studies of patients with localized (Stage 0 or 1) lung cancer,however, 5-year survival rates have ranged from 40% to 70% (Boring &Squires, 1993, supra; Ferguson, 1990, supra).

Historically, the only diagnostic tests used to detect lung cancerbefore symptoms occur have been sputum cytology and chest radiography.As a consequence, the efficacy of these tests as mass screening toolshas been extensively evaluated in studies over the past several decades.Both tests detect presymptomatic, earlier-stage carcinoma, particularlycarcinoma of squamous cells.

Improvements in screening methods have largely centered around improvingthe utility of sputum cytology through technologic advances inmicroscopy. Sputum cytology requires a visual examination of a cellsample during which cell size, shape, organization, and a ratio betweenthe size of the cell's nucleus and cytoplasm is used to determine thecell's morphology. Because this assessment of cell morphology requires avisual inspection and classification, the technique requires asignificant amount of expertise on behalf of the clinical observer.Various investigations have been conducted with results suggesting thatcomputer-assisted, high resolution image analysis enables detection ofsubvisual changes in visually normal nuclei associated with severaltissue types (Montag et al. 1991, Anal Quant Cytol Histol 13:159-167;Haroske et al. 1988, Arch Geschwulstforsch, 58:159-168; Hutchinson etal. 1992, Anal Quant Cytol Estol 4:330-334). Computer-assisted analysisof DNA distribution in cell samples provided 74% correct morphologicalclassification of nuclei without human review of the material andwithout the need for visually abnormal nuclei being present whencompared with standard cytological testing.

The morphologic assessment of cytological specimens has also improveddue to advances in the understanding of lung tumor pathology. Much ofthis work has centered on the identification of “biomarkers.” Biomarkersrefer to a wide range of progressive phenotypic and geneticabnormalities of the respiratory mucosa which may be used in determiningthe potential for bronchial epithelium to fully transform into amalignant tumor. Markers have been broadly classified as morphologicalchanges, immuno/histochemical markers for differentially expressedproteins, markers for genomic instability, markers of epigenetic change(e.g., abnormal methylation), and gene mutations (Hirsh et al. 1997,Lung Cancer 17:163-174).

The expression levels of these markers are now being evaluated indysplastic and neoplastic cyto/histological tissue samples collectedfrom high risk populations. Among those specimens currently beingtargeted for exploratory marker analysis is sputum. Interest in sputumsamples for biomarker research has been generated from the long-heldbelief that exfoliated cells recovered in sputum may be the earliestpossible indication of an incipient carcinoma, since lung cancer mostfrequently develops in the bronchial epithelium. Through application ofsophisticated molecular genetic techniques (e.g., PCR-based assays),studies are providing evidence that selected biomarkers can be detectedin sputum (Mao et al. 1994, Cancer Res 54:1634-1637; Mao et al. 1994,Proc Natl Acad Sci USA 91:9871-9875; Sidransky 1995, J Natl Cancer Inst87:1201-1202; Tockman et al. 1988, J Clin Oncol, 11: 1685-1693; Tockmanet al. 1994, Chest, 106:385s-390s).

Commercially available cancer screening or detection services rely ontests based on cytomorphological diagnosis by trained clinicians wholook at each sample and determine the extent and identity of abnormalcell types. This process is not only expensive and time-consuming, italso introduces human judgment and therefore error into the procedure.Recently, a method has been developed for detecting cancerous cells ofthe lung through use of 5,10,15,20-tetrakis (carboxyphenyl)-porphine(TCPP) (U.S. Pat. No. 5,162,231 to Cole et al.) This method relies onthe propensity of cancerous cells to accumulate TCPP from theirenvironment in a greater amount than non-cancerous cells. Uponincubation of a cell sample for 6-24 hours with 200 μg/ml TCPP, the TCPPentered cells and bound to the perinuclear membrane and mitochondria ofneoplastic cells. TCPP fluoresces under ultraviolet light, and cancerouscells may thereby be diagnosed solely by the intensity of fluorescence,without reference to morphology. The extension of the use of thiscompound to identifying pre-cancerous tissue conditions (e.g.,dysplastic cells) would permit screening in high risk populations toidentify those individuals whose tissues are progressing toward invasivecancer conditions, and thereby facilitate catching the cancer ordysplasia at the most treatable stage. The desirable characteristics ofsuch a screening method would be a procedure that is rapid, inexpensive,and requires a minimum of technical expertise.

For the foregoing reasons, there is a need for a technique andmethodology for detecting dysplastic cells in their earliest stages. Inaddition, there is a need for a technique that can provide highlyreliable diagnostic results and that does not rely on the subjectiveanalysis of the clinician performing the diagnosis.

SUMMARY OF THE INVENTION

The invention is derived from the discovery that TCPP can be used todetect dysplastic and precancerous as well as cancerous cells, inconjunction with a novel and more efficient method of solubilizing TCPP,improved staining procedures, and a variety of cell sorting strategies.TCPP is a fluorescent compound that has now been discovered to bind tocomponents of live or fixed precancerous as well as cancerous cells in amanner that allows the state of the cells and the tissue from which theycame to be categorized on a disease progression continuum. This methodof detection of precancerous tissues is well-suited to in vitrodiagnosis of tissue or cell samples as well as in situ diagnosis.

One aspect of the invention is a method for detecting precancerouscells, which in its simplest form comprises incubating live or fixed(i.e., killed) cells in a TCPP solution for sufficient time to bind tothe cellular components, and detecting the bound TCPP with fluorimetry.This method has many variations. In one variation, the cells are fixedon a surface, preferably a microscope slide, and most preferably in amonolayer. In another variation, the cells are treated with formalin oranother suitable fixative solution, maintained in suspension, treatedwith TCPP, the cells separated from the unbound TCPP, and then analyzedand sorted by flow cytometry.

Preferred embodiments of the incubation step include using a TCPPsolution with about 4 μg/mL to 400 μg/mL TCPP, a temperature betweenabout 23° C. and about 42° C., and a time between about 0.2 minutes to 2hours. Unbound TCPP is removed and the remaining TCPP is detectedfluorimetrically. In a preferred embodiment, the TCPP is detectedbetween about 1 and 24 hours after the assay is performed.

In another embodiment of the invention, the percentage of fluorescentcells in a cell sample is calculated. Preferred embodiments compriseanalysis of fluorescent cells for their fluorescence intensity and othercytomorphological features. In a particularly preferred embodiment,fluorescent cells are classified according to a set of pre-determinedfluorescence intensity and cytomorphological features, which facilitatescharacterization of the cells along a continuum ranging from normal tometaplastic to dysplastic (mildly to severely) to carcinomic (mildly toseverely), and increases the efficiency and reliability of the diagnosesand prognoses made using the methods of the invention. Other embodimentsof the invention comprise separating the normal or metaplastic cells ina sample from the dysplastic or carcinomic cells, using criteria offluorescence intensity (e.g., via fluorometric flow cytometry).

In order to facilitate practice of the aforementioned detection method,another aspect of the present invention provides a method for making aTCPP solution comprising dissolving TCPP in about 50% to about 90%alcohol at a pH greater than about pH 8.5 and less than about pH 12.5.In one preferred embodiment, the alcohol is isopropanol, and in anotherpreferred embodiment the pH of the solution is adjusted with sodiumbicarbonate or ammonium hydroxide.

Another aspect of the invention is a composition that comprises TCPP inabout 50% to about 90% alcohol at a pH greater than about pH 8.5 andless than about pH 12. In one preferred embodiment, the alcohol isisopropanol, and in another preferred embodiment the pH of the solutionis adjusted with sodium bicarbonate or ammonium hydroxide. In oneembodiment, the composition is made by the method for making a TCPPsolution. In a related embodiment, the TCPP solution used in thedetection method is diluted.

In another aspect of the invention, cells are in situ within a mammalianpatient. Cells are exposed to TCPP solution and fluorescence is detectedby using endoscopic techniques.

Another aspect of the invention is a kit for detecting precancerouscells, which comprises the composition of the invention in a container.In another embodiment, the kit comprises one or more additionalcomponents, such as instructions and reagents for carrying out thedetection method of the invention, or positive and negative controls.

Other features and advantages of the present invention will be betterunderstood by reference to the drawings, detailed description andexamples that follow.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises compositions and methods for detectingprecancerous conditions in human cells using 5,10,15,20-tetrakis(carboxyphenyl) porphine (TCPP). The present invention derives from thediscovery that TCPP binds specifically to precancerous abnormal cells inaddition to cancerous cells, but does not bind to normal (noncancerous)cells. Moreover, this differential binding is observed in fixed cells aswell as live cells. In addition to this new and useful property of TCPP,the invention additionally incorporates an improved method forsolubilizing TCPP that preserves its activity to a greater extent, aswell as several novel aspects that make the method better suited toscreening and automation. Using the methods of the invention, less timeis required for the cells to bind the TCPP when compared with the methoddescribed in U.S. Pat. No. 5,162,231 (e.g; 0.2 min-2 hours versus 24hours), and a lower concentration of TCPP (e.g., 40 μg/mL versus 200μg/mL) is also required. In addition, a monolayer of cells may be usedrather than a solution of cells, though a solution of cells may also beused.

Key to the convenience and efficiency of the detection method is thenovel method of solubilizing the TCPP. Previous methods used 1 M NaOH todissolve the porphyrin. That method required titration with 1M HC1,which is inaccurate, and required that each solution be checked forundissolved TCPP. Additionally, the NaOH method placed the porphyrin inan oxidizing environment with pH as high as 13.0, thereby exposing theporphyrin to a high risk of degradation. The solubilization method ofthe present invention uses pH 9.1 in conjunction with the novel additionof 90% alcohol to effect a more complete and reliable solubilization.Finally, the method of this invention uses a buffer to stabilize the pHof the final working TCPP solution in the preferred range of 5.8 to 6.8.

The invention involves the detection of precancerous and cancerous cellsin samples of human tissues using the unique propensity of these cellsto bind TCPP in a greater amount than healthy cells. As used herein, theterms “precancerous” or “abnormal precancerous” refer to cells thatexhibit mild to severe dysplasia, and the term “cancerous” refers tocells that exhibit mild to severe carcinoma. These cytological statesare morphologically defined herein by the criteria used to determinecell morphology using Papanicalou-stained (“PAP-stain”) cytology. Theyalso may be defined by other indicators commonly used in the art for aparticular cell or tissue (e.g., indicators of pulmonary inflammation inlung or sputum samples). From “normal” to “severely carcinomic,” thestates of a cell are classified herein as (1) normal (no significantabnormalities), (2) metaplasia (squamous metaplasia), (3) mild dysplasia(squamous atypia), (4) moderate dysplasia (squamous atypia), (5) severedysplasia (marked squamous atypia), (6) carcinoma in situ squamous (CIS,non-invasive) (also referred to as mild to moderate carcinoma), and (7)squamous cell carcinoma (well-differentiating keratinizingtype-invasive) (also referred to as moderate to severe carcinoma). Ithas been determined in accordance with the invention that followingexposure to TCPP, dysplastic and carcinomic cells display TCPPfluorescence, while normal cells display little or no TCPP fluorescence.Some metaplastic cells may display low to moderate TCPP fluorescence,but in many instances they do not; hence TCPP fluorescence is not asreliable an indicator for metaplasia as it is for dysplasia andcarcinoma.

The method comprises (1) incubating a sample of fixed or living cellswith TCPP for a time sufficient to allow TCPP to bind to cellularcomponents of abnormal precancerous or cancerous cells, if any arepresent in the sample, (2) removing unincorporated TCPP, (3) determiningby fluorimetry the amount of TCPP remaining in the sample, if any; and,optionally, depending on the results of step (3), (4) evaluating TCPPfluorescing cells for their state of divergence toward cancer from thenormal (or abnormal metaplastic, but not dysplastic), state.Specifically, as described above, the method of the invention enables adetermination that a cell sample contains cells which are dysplastic(mildly to severely) or carcinomic (mildly to severely).

In an exemplary, but non-limiting embodiment, the detection methodcomprises the following steps:

-   -   1. fixing cells in a monolayer on a microscope slide;    -   2. exposing cells to TCPP solution at about 40 μg/mL in a        buffered solution at about pH 6.1 (e.g., by dipping slides into        the solution or by placing drops of solution onto the slides) at        about 36° C. for a specified time, as described below;    -   3. washing slides with a buffered solution at about pH 6.1;    -   4. waiting at least 1 hour but not more than 24 hours; and    -   5. quantitating fluorescence from cells at about 610-740 nm when        excited with about 380-450 nm light.        Variations of this exemplary method are set forth in greater        detail below.

When used herein in describing components of assay mixtures or otherparameters of the invention, the term “about” means within a margin ofcommonly acceptable error for the determination being made, usingstandard methods.

The first step, incubating the cells with fixative, is optional, butpreferred, inasmuch as it has been found to reduce the time required forincubation, as well as the TCPP concentration in the working solution,in this exemplary embodiment and in others.

The sections below set forth a variety of other embodiments of thepresent invention.

The methods of the invention may be used on a variety of cell types asdescribed below and further are applicable to veterinary as well ashuman diagnostic and prognostic applications. Accordingly, the term“patient” or “subject” as used herein is intended to apply to a human oran animal.

The detection method may be used to detect precancerous and cancerouscells in cell samples in vitro. Cell samples may be acquired by any ofthe methods currently used in the field of cytopathology. For example,cells may be collected from sputum samples (see Example 1), cervicalswabs, bronchial washings, fine needle aspiration and core biopsies ofthyroid and breast, bladder washings, urine, mouth washing, enemas, andother biopsies known in the art. Other sources of cell samples includeblood or fractions thereof, lymph, cerebrospinal fluid, bone and bonemarrow, to name a few. The method of the invention is applicable to anycell sample from any tissue or organ in the body.

Optionally, the cells may be fixed by standard procedures beforeexposure to TCPP, including but not limited to solutions containingformaldehyde, methanol, ethanol, or isopropanol. In one embodiment, thecells are fixed in 95% ethanol.

The assay may be performed in solution by measuring total fluorescenceper cell density, or by adhering the cells onto a surface. Cells neednot be treated with a fixative, but fixing cells is preferred in someembodiments, particularly those in which the cells are adhered to asolid support. In one embodiment, the cells are adhered as a monolayerto a slide. In other embodiments, the liquid-based slide preparationsystem MonoPrep2 or MonoPrepG (MonoGen, Inc., Herndon, Va.) or theThinprep Processor (Cytyc Corporation, Marlborough, Mass.) is used.

The method of the invention may also be used to detect precancerous andcancerous cells in situ as well as an aid in resective surgery. Forexample, the method may be used to detect dysplastic cells in the lungin situ by injection of TCPP in a suitable medium followed byfluorescence bronchoscopy. A similar method may also be used to detectabnormal cells for excision during surgery. In situ applications may befound for any of the organs of the body, including, but not limited to,breast, prostate, lungs, cervix, throat, bladder, oropharynx, skin, andgastrointestinal tract by use of a similar endoscopic device. The amountof TCPP preferred for use in this embodiment is determined by the modeof administration and the site of delivery. For instance, if TCPP isinjected into the bloodstream, the effective concentration of TCPP willdepend on its maximum solubility in saline or blood (e.g., about 100μg/mL). For injection directly into affected tissue, an effective amountof TCPP will depend on the target tissue and the proximity of theinjection to that tissue (e.g., about 1-20 mg). In lung, an aerosoldelivery of, e.g., 5-10 ml at a concentration of 20-50 μg/mL should besuitable. Methods of determining such amounts of TCPP to be administeredas a diagnostic agent are well known to medicinal chemists and others ofskill in the relevant art.

The TCPP concentration in the working solution and the duration of timethe cells are exposed to TCPP solution are two variables that may bealtered in a coordinated manner. The TCPP concentration is preferably4-100 μg/mL, more preferably 4-40 μg/mL, and most preferably 20-40μg/mL. The time of exposure may vary from about 0.2 minute to about 2hours in one embodiment, and 10 to 60 minutes in a more preferredembodiment. When low concentrations of TCPP solution are used, a longerexposure time is appropriate, and when high concentrations of TCPP areused, a shorter exposure time is appropriate. For instance, in preferredembodiments, slides are exposed for 10 minutes in 40 μg/mL TCPP, andalternatively for 60 minutes in 4 μg/mL TCPP. The method to optimize theconcentration of TCPP in the working solution and time of exposure arewell known to those skilled in the art of cytology, the goal being toachieve the highest specific binding of TCPP to the cellular componentswhile minimizing the background and other non-specific uptake andfluorescence.

The TCPP solution is comprised of TCPP in a buffered aqueous medium atabout 36° C. In one embodiment, the buffering capacity is due to 100 nMMES; however, a concentration range of 20 to 200 mM can be used in themethod with equal efficacy. In one embodiment, the solution has a pH ofabout 6.1; however, a pH range from 5.8 to 6.7 may be used withsufficient efficacy. Other buffering compounds that are effective in therange of pH 5.8-6.7 may also be used. While the exposure step is notparticularly sensitive to temperature, a temperature somewhat above roomtemperature is desirable for optimization. The suitable temperaturerange for the exposure step is about 23° C. to about 42° C. in apreferred embodiment and about 30° C. to about 40° C. in a morepreferred embodiment.

Other compounds may be added to the working solution to reducebackground fluorescence, increase stability, or reduce autofluorescenceor quenching. For example, detergents may be used to lower backgroundfluorescence and reductants, antioxidants, and other inhibitors of thegeneration or diffusion of active oxygen species may be used to preventoxidation of the TCPP or reduce photobleaching. Compounds of interestinclude, but are not limited to, polyethlyne glycols, tritons,dithiothreitol, dithiocrithritot, 2-mercaptoethanol, or the “Antifadekits” supplied by Molecular Probes, Inc. (Eugene, Oreg., P-7481, S-2928,S-7461). In addition, hematoxylin may be included with the TCPP, to actas a counterstain and facilitate white light microscopy.

The wash solution is generally similar to the aqueous solution used forthe TCPP working solution but without the TCPP. If microscope slides areused, the slides should be washed at least once, most preferably threetimes, and preferably with agitation in excess wash solution. If theassay is done in solution, unbound TCPP may be removed by centrifugingthe cells, decanting the supernatant and resuspending the cells in freshbuffer. This step may be repeated if necessary. Alternative means ofseparating cells from staining solution may also be used, such asfiltration with capture of cells on a membrane or rapid dialysis(including spin dialysis). The proper conditions of the wash may bedetermined by monitoring the fluorescence of the cells. The wash shouldbe sufficient to remove background and other non-specific binding, butnot so excessive as to remove specifically bound TCPP from the cellularcomponents. The optimization of the wash step is well known to those inthe art of cytology. Compositions that may be added to the wash solutionto improve efficacy or stability include, but are not limited to,alcohols, detergents or low molarity salt solutions.

An important step in the detection method is to allow more than 1 hourbut less than 24 hours between the time the slide has been exposed tothe TCPP working solution to the time the slide is read. When the slideis read after 24 hours, there may be too much deterioration in the TCPPto yield an accurate result. When cells are read before 1 hour, thelevel of background fluorescence may be excessively high.

The wavelengths used for the detection step are encompassed by broadranges in which specific peak ranges will be most efficient. The peak ofexcitation of TCPP in an aqueous solution of pH 5.0-7.0 occurs at about415 nm, while one peak of emission occurs at about 645 nm and asecondary peak of emission occurs at about 706 nm. In general, TCPP maybe detected by illuminating the sample with ultraviolet (UV) light anddetecting the light emitted from the sample above about 500 nm. Thewavelengths used to excite TCPP in the method of the invention maypreferably encompass part or all of the range from about 380 to about450 nm, and most preferably a narrow band of wavelengths near about 415nm. Likewise, the wavelengths detected may encompass part or all of therange from about 610 nm to about 740 nm and most preferably a narrowrange near about 650 nm. Under certain circumstances obvious to thoseskilled in the art of fluorescence microscopy, it may be preferable todetect emissions in a narrow range near about 706 nm. The selection ofwavelength can most easily be accomplished by optical filters. Filtersets for popular fluorescent dyes are readily available from MolecularProbes (Eugene, Oreg.), for example. The appropriate wavelengthselection to excite and detect TCPP in the method of the invention maymost easily be obtained by using a filter set designed for detectingfluorescein isothiocyanate (hereinafter “FITC”), which generally has anexcitation filter of 400 to 490 nm and a barrier filter for emissionabove 500 nm. The selection of other filter systems is well known tothose skilled in the art of microscopy.

Fluorescence may be detected visually or mechanically, manually or byautomated means. Cells having even moderate fluorescence as compared tonon-fluorescing cells are easily distinguishable by the human eye.Hence, certain embodiments of the invention comprise simply viewingTCPP-treated cells under a fluorescence microscope and quantitating thepercentage of fluorescing cells in the sample manually. However,preferred embodiments comprise automated methods well known in the art,and mechanical quantitation wherein a cell is “counted” as fluorescentif it displays fluorescence over a pre-determined threshold levelprogrammed into the counting device, as is well known in the art. Forinstance, in embodiments comprising a monolayer of cells adhered to amicroscope slide, an automated slide reader can be programmed to countas fluorescent any cell having fluorescence pre-determined to be ofstatistical significance compared to an equivalent, normal cell, asdetermined by standard statistical methods (such devices also may beprogrammed to count cells of a certain shape, which can supply a secondindicator of precancerous or cancerous abnormality). Alternatively, forembodiments comprising a solution-based assay, TCPP-stained and washedcell samples may be analyzed by fluorescence-activated cell-sorting(FACS), wherein the FACS is programmed to separate cells having apre-determined level of fluorescence, as may be determined statisticallyby comparison with normal cells.

The total number of cells present in a sample is determined in order tocalculate a percentage of that total that are TCPP-fluorescent. Thisdetermination may be accomplished in a variety of ways known in the art.In one embodiment all cells are stained with hematoxylin and countedunder white light microscopy. In another embodiment, the cells arestained with a suitable fluorescent counter stain (e.g., one that stainsthe external or internal membranes of a cell) that fluoresces at adifferent wavelength from TCPP. In this latter embodiment, the ratio ofTCPP fluorescence to cell marker fluorescence is quantitated.

As mentioned, the novelty of the methods of the invention resides in theinventors appreciation that TCPP staining identifies not only cancerouscells, as previously known, but also identifies precancerous dysplasticcells. Because of this, the above-described method yields vastly moreinformation than previously believed possible. Accordingly, the resultsof the TCPP fluorescence quantitation will be determinative of whethersubsequent analytical steps are taken, and in what form.

For instance, the method will identify a percentage of cells in a samplethat are TCPP fluorescent. If about 1-3%, more particularly about 2-3%,of the cells in a sample are fluorescent, then sample contains cellsthat are at least abnormal precancerous (dysplastic) or cancerous.Accordingly, a simple analytical scheme involves determining if a samplecontains at least about 1% TCPP-fluorescent cells. If it does not, thesample is diagnosed as negative (normal). If it does, further testing isrecommended for the patient. It should be noted in connection with thisembodiment that, even if a sample contains fewer than 1% fluorescentcells, other factors (e.g., pre-disposition of the patient to cancer, ora pre-existing cancer in another tissue) may suggest that furthertesting be performed. One advantage of the invention that is describedin greater detail below is that an enriched population of fluorescentcells may be obtained from the patient via FACS.

In addition, the level of fluorescence of a given cell in a sample hasbeen found to correlate with the cancer-associated state of that cell(see Example 1). Accordingly, individual cells or groups of cells may beevaluated for their overall fluorescence intensity, and a determinationof whether further testing is required may be based in part on thisevaluation.

The terms, “high”, “medium” and “low” fluorescence and related terms asused herein, will be understood by one of skill in the art to becomparative terms wherein the fluorescence intensity of a single cell orgroup of cells in a test sample is compared at least with cells from anequivalent source (e.g., sputum) known to be normal with respect tocancer (negative control). This comparison may be accomplished by visualestimation, or, in automated systems, it may be programmed usingstatistical parameters such as variation from the median fluorescence ofa sample population, as described in Example 2. In preferredembodiments, cells from a test sample are compared for fluorescenceintensity with additional control cells whose cancerous state has beenpre-determined and pre-correlated with a TCPP fluorescence intensity(for instance, as described in Example 1).

The terms “fluorescent” and “non-fluorescent” are also used herein. Inkeeping with the above-discussed definitions of various levels offluorescence intensity, the terms “non-fluorescent” and “fluorescent”are used as comparative terms, wherein fluorescence is compared againstnormal cells from an equivalent source, and/or against generalbackground fluorescence arising from the reagents or equipment used indetecting the fluorescence. Hence, if a cell or cell sample isdetermined “fluorescent,” then fluorescence is present at some intensityabove background fluorescence of fluorescence observed in known normalcells. If a cell or cell sample is determined “non-fluorescent,” thenthe fluorescence observed is minimally or not at all in excess ofbackground fluorescence or fluorescence observed in normal cells. Thiscomparison would be understood by one of skill in the relevant art.

A cytomorphological evaluation combined with TCPP fluorescence isparticularly useful with cell cultures that have a low level offluorescence because a visual evaluation of the cells with standardevaluation techniques can easily differentiate the slightly fluorescingmetaplastic (non-cancerous) cell from dysplastic (precancerous) cells.One embodiment of the method comprises an additional step ofcytomorphological evaluation in addition to quantitation offluorescence, especially using a standard cytological stain such ashaematoxylin to help visualize cell and nuclear outlines. Anotherembodiment employs cytomorphological evaluation as a subsequent step, ifcertain threshold requirements are met, e.g., the sample contains morethan 1% fluorescent cells.

In a particularly preferred embodiment of the invention, selectedcytomorphological features are combined with fluorescence intensity toproduce a classification system that is very useful for efficient,reproducible diagnosis of the various stages of metaplasia, dysplasiaand carcinoma that may be present in a cell sample. Such aclassification system is described in detail in Example 1. In thisembodiment, TCPP-fluorescent cells are assigned one or more numericalclasses, based on fluorescence intensity and simple morphologicalfeatures including cell shape and size, number or size of nuclei,presence of cell clusters and degeneration of cells or cell clusters,presence of irregular anisoid cells, visibility of cell membrane, andpresence and nature of nuclear debris. The technician or scientistperforming the cytomorphological evaluation of TCPP-fluorescing cellscan use the classification as a checklist, i.e., a cell being examinedmay be checked off as “plus” or “minus” with respect to each of thenumerical classes. The number of numerical classes assigned to aparticular cell and the pattern of specific classes assigned to a cellare both informative as to the cancerous or precancerous condition ofthat cell. By way of illustration, Example 1 sets forth a classificationsystem comprising 14 numerical classes. As shown in Table 2 of thatexample, which assays presents of sputum samples, negative ormetaplastic cells generally are assignable to few of the classes, whileseverely carcinomic cells are assignable to several. As furtherillustration, negative or metaplastic cells are frequently assignedclass 11, while moderately dysplastic to carcinomic cells are not, andcarcinomic cells are frequently assigned class 6, while normal,metaplastic and dysplastic cells are not.

In another embodiment of the invention, TCPP-treated cells in solutionfrom a single patient determined to have carcinoma can be separated byflow cytometry based on their level of fluorescence. Cells showing ahigher level of fluorescence are considered to be cancerous while cellswith moderate to low levels of fluorescence are considered dysplastic,and cells with no fluorescence are considered to be normal. This type ofseparation enables a patient's dysplastic or cancerous cells to becompared against the patient's own normal cells, thereby providing anideal “internal” control population.

In another embodiment, by separating cancerous and normal cells from thesame patient, various chemotherapeutic agents can be assayed to test foreffectiveness. Separated cells are dispensed into aliquots. A selectedtherapeutic agent can then be mixed at the same concentration with analiquot of highly fluorescent cells and an aliquot of low levelfluorescent cells. This step can be repeated with fresh aliquots and adifferent therapeutic agent. Cell death rates can be assessed usingtechniques known in the art. The most preferred therapeutic agent fortreatment can then be determined by choosing the chemo-therapeutic agentthat killed the highest number of cells determined to be cancerous(i.e., highly fluorescent cells) and killed the fewest number of normalcells (i.e., cells with little or no fluorescence after TCPP treatment).

In conjunction with the screening or diagnostic detection method of theinvention, a method for dissolving TCPP for use in the method as well asother applications has been developed. This method comprises dissolvingTCPP in about 50% to about 90% alcohol with a pH greater than about pH8.5 and less than about pH 12.5. Preferred for use in the invention arelower alcohols such as methanol, ethanol, isopropanol and n-propanol.More preferably, the alcohol is isopropanol and its pH is adjusted withsodium bicarbonate or ammonium hydroxide to a pH greater than 8.5 andless than 10.0. The concentration of isopropanol may be from 50% to 90%and the sodium bicarbonate may be from 20 mM to 100 mM in someembodiments. The concentration of TCPP may be up to about 2 mg/mL. Inone embodiment, the TCPP is dissolved at 1 mg/mL in a solution of 50%isopropanol 50 mM sodium bicarbonate.

The invention also comprises a composition useful for use in any methodutilizing TCPP that comprises TCPP in alcohol with a pH greater than 7.This solution would preferably be made by the method for dissolving TCPPdetailed above. This composition should preferably be stored at about 4°C. in the dark.

The invention additionally encompasses kits for the detection ofprecancerous and cancerous cells comprising TCPP in a container,optionally with instructions. In one embodiment, the kit is designed tobe used with the detection method of the invention. In one embodiment,the kit includes the composition of the invention comprising TCPPsolubilized in basified alcohol in a container. This TCPP solution maybe used as a stock solution which would be diluted into a bufferedaqueous solution for the purpose of detecting precancerous and cancerouscells. The kit may contain the components for collecting the cellsample, as in the sputum collection container of Example 1, oralternately may contain items for the detection of precancerous cells insamples already acquired. The kit may be tailored for use with slidepreparation systems, e.g., MonoPrep2 or MonoPrepG (Monogen, Inc.,Herndon Va.) or the Thinprep Processor (Cytyc Corporation, Marlborough,Mass.), to name three. These kits may also be designed to be used withformats other than microscope slides, such as microtiter plates or flowcytometry devices. In any of the foregoing embodiments, the kit maycomprise positive or negative controls, or both, as would be employed byone of skill in the art in conducting the assays of the invention.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

EXAMPLE 1 Glass Slide Assay for Detection of Pre-Cancerous and CancerousCells with TCPP

This example compares the diagnostic results achieved through standardcytomorphologic analysis of PAP-stained sputum slides versus slidestreated with TCPP and analyzed by fluorescence microscopy. The resultsindicate that the TCPP detection technique of this invention isequivalent to conventional sputum cytology in the detection ofneoplastic cells (dysplasia and carcinoma in situ) and frank carcinomas.The results also indicate that one skilled in the art can use themethod, in conjunction with simple classification rules, to estimate thedegree of dysplasia present in a tissue sample.

Methods

Sputum Processing Procedures Used in Production of Monolayer Slides. Allmonolayer slides selected for analysis in this study were produced fromsputum samples collected through patients performing the early morning,spontaneous cough technique. Specifically, patients were instructed toexpectorate whatever material they coughed up across three consecutivemornings into a container filled with fixative consisting of 2% Carbowaxin 50% alcohol/50% Saccomanno fluid with 0.03-0.05 mg/mL rifampin.Rifampin was added to the fixative solution to serve as a prophylacticagainst patients harboring M. tuberculosis or those patients who may beasymptomatic carriers of N. meningitis.

The 2% Carbowax solution was prepared by adding 2 mL melted Carbowax(150) to 98 mL of 50% ethanol and mixing for 30 minutes. Glassware usedto make solution was kept warm to prevent hardening of wax on thesurface during preparation, which can cause inaccurate measurement.Carbowax was removed before exposure to the TCPP working solution byimmersion in 95% alcohol for at least 15 minutes.

Rifampin solution (3 mg/mL) was made by dissolving 300 mg capsules ofrifampin into 100 mL ethyl alcohol and blending at high speed in aWaring blender. One mL of this solution was added to each 30 mL ofSaccomanno solution or 20 mL per liter of Saccomanno solution and mixedthoroughly. The preparation of Saccomanno solution was according tostandard methods well known to those in the art of cytology.

Two thin-prep microscope slides (Cytyc Corporation, Marlborough, Mass.)and a 50 mL plastic centrifuge tube were labeled with patientinformation. The sputum specimen was poured into a 50 mL plasticcentrifuge tube and additional 50% ethyl alcohol solution added to bringthe volume to 50 mL if necessary. The contents of the centrifuge tubewere poured into a 250 mL Eberbach semi-microblender container andhomogenized for 10 to 60 seconds, depending upon visual examination ofthe specimen and mucoid content. Thick mucoid specimens sometimesrequired longer blending times. The specimen was poured back into thecentrifuge tube and centrifuged at 1850 rpm for 10 minutes. Thesupernatant was decanted, leaving 1 to 2 mL in the centrifuge tube toadmix with the sediment (centrifugate). The tube was agitated on avortex mixer for approximately 10 seconds. One to three drops of thesediment was placed into a PreservCyt vial (Cytyc Corporation,Marlborough, Mass.). The specimen was incubated for 5 minutes todeactivate all microbial and viral organisms.

Monocellular layers of the samples were fixed onto slides using theThinPrep Processor (Cytyc Corporation, Marlborough, Mass.) according tothe manufacturer's instructions. In the ThinPrep Processor, cells werecollected onto a polycarbonate filter (pore size 0.5 mm) and transferredto a glass slide. The ThinPrep Processor then immediately deposited theslide into a fixative bath containing 95% ethanol.

TCPP Stock Solution. 400 mg of sodium bicarbonate was added toapproximately 90 mL of the 50% isopropanol (50 mM sodium bicarbonate)and mixed until completely dissolved to make basified 50% isopropanol.One hundred milligrams of TCPP were slowly added to the basified 50%isopropanol (50 mM sodium bicarbonate) and mixed for 3 to 5 minutesuntil dissolved. The TCPP solution was brought to 100 mL volumetricallywith the basified 50% isopropanol, mixed well and stored in an amberreagent bottle covered in foil in a refrigerated area. The finalconcentration of TCPP in the stock solution was 1 mg/mL.

TCPP Working Solution. Fresh TCCP working solution was prepared eachday. Approximately 10 mL of TCPP Stock Solution with concentration 1mg/mL was brought to room temperature. Eight milliliters of the TCPPStock Solution (1 mg/mL) were placed in a 200 mL volumetric flask andapproximately 100 mL of the MES buffer was slowly added. The solutionwas gently mixed. Additional MES buffer was added to bring the solutionto 200 mL volumetrically. The solution was mixed for 3 to 5 minutes andstored at 2-4° C. in an amber bottle. The final concentration of TCPP inthe working solution was 40 μg/mL.

TCPP Exposure Procedure. Slides were fixed in 95% alcohol for 30 minutesat room temperature. The slides were exposed to TCPP immediately afterfixing or up to 3 days later. The slides were immersed in 40 μg/mL TCPPsolution for 10 minutes at 36° C., then washed in 100 mM MES bufferthree times, one minute each, at room temperature with agitation. Slideswere viewed more than 1 hour but not more than 24 hours later.

Microscope Information. The microscope utilized for observation of theTCPP treated sputum cell slides was an Olympus model BH-1 microscopewith a sub-stage illuminator and above-stage mercury lamp for reflectedlight fluorescence microscopy. The mercury lamp has primary emissionlines at 365 nm, 405 nm, 436 nm, and 545 mm. The fluorescence filterassembly consisted of two dichrotic cubes. The green cube (490 nm)contained a filter system with an excitation filter passing 400-490 nmand a barrier filter passing emission above 500 nm.

Sputum Slide Staining Procedures by Modified PAP-Staining Technique.Procedure sequence (no.), reagent and time (min.:sec.) were as follows:(1) 95% alcohol 15:00; (2) tap water 1:00; (3) gil-i hemotox 2:30; (4)tap water 1:00; (5) bluing reagent: 30; (6) tap water 1:00; (7) 95%alcohol: 10; (8) og-6 1:30; (9) 95% alcohol: 10; (10) 95% alcohol: 10;(11) ea-50 1:15; (12) 95% alcohol: 20; (13) 95% alcohol: 30; (14) 100%alcohol 1:00; (15) 100% alcohol 1:00; (16) 100%-alcohol 1:30; (17)xylene 1:00; (18) xylene 1:00; and (19) xylene 1:00.

Methods for Routine Cytopathological Analysis of Papanicolaou-StainedSlides. PAP-stained slides underwent semi-quantitative cytomorphologicalevaluation. (1) dysplastic and neoplastic cells were identified throughuse of traditional morphologic criteria, and (2) the expression levelsof seven fundamental indicators of pulmonary inflammation (alveolarmacrophages, neutrophils, columnar cells, mucus, mucous spirals,pigmented macrophages, metaplastic cells) were quantified. Themethodology for quantifying these inflammation indicators have beenpreviously discussed in the literature (Roby et al., 1989, Acta Cytol34:147-154; Roby et al., 1990, Acta Cytol 34:140-146; Schumann et al.,1989, Am Rev Respr Dis 139:601-603). The criteria used to determine cellmorphology using PAP-stained cytology are discussed below.

No significant abnormalities. Cells were identified as having nosignificant abnormalities if the following criteria were satisfied:

-   -   1. basophilic, ciliated epithelial cells admixed with        macrophages with grade 1-2 pigment along with inflammatory        cells;    -   2. round nuclei of epithelium basally oriented;    -   3. evenly dispersed chromatin;    -   4. inconspicuous nuclear membranes;    -   5. inconspicuous nucleoli; and    -   6. no metaplastic and no dysplastic cells present.

Squamous metaplasia (without dysplasia). Cells were identified assquamous metaplastic without dysplasia if the following criteria weresatisfied:

-   -   1. clumps of basophilic cells without cilia;    -   2. uniform cell and nuclear size;    -   3. low nucleus/cytoplasm (N/C) ratio;    -   4. nuclear chromatin finely granular; and    -   5. small rounded nucleoli (usually single) may be present.

Mild dysplasia (squamous atypia). Cells were identified as mildlydysplastic if the following criteria were satisfied:

-   -   1. smaller than metaplastic cells;    -   2. seen in cohesive clusters, or singularly;    -   3. cells lie flat (sheets) both nuclei and cytoplasm in focus;    -   4. cells vary slightly in size;    -   5. cytoplasm may be eosinophilic or basophilic;    -   6. cytoplasmic borders sharp;    -   7. nuclei vary slightly in size, usually round to oval, if        divided 2 halves of nucleus are mirror images, N/C ratio may        vary slightly;    -   8. nuclear membrane smooth;    -   9. nuclear chromatin (slightly increased) finely granular,        occasional chromocenter; and    -   10. fiber cells, elongated cells with stretched cytoplasm and        nucleus distinct nuclear membrane—fine reticular to granular        cytoplasm usually bright yellow orange—keratinizing single, may        form whorls around central core of keratin to make epithelial        pearls.

Moderate dysplasia (squamous atypia). Cells were identified asmoderately dysplastic if the following criteria were satisfied:

-   -   1. variation in size, usually larger but may be smaller than        mild dysplasia;    -   2. more variation in shape and N/C ratio than mild dysplasia;    -   3. cytoplasm dense, acidophilia predominates; increased number        of atypical cells;    -   4. nucleus may have unequal halves (not mirror images);    -   5. nuclear lobulations, crevices, and nodules are present; and    -   6. nuclear material may show hyperchromasia with more        stippled—like chromatin pattern.

Severe dysplasia (marked squamous atypia). Cells were identified asseverely dysplastic if the following criteria were satisfied:

-   -   1. cells vary markedly in size and shape;    -   2. usually slightly larger cell size than moderate dysplasia;    -   3. N/C ratio is high but variable (with extremes);    -   4. single cells predominate; nucleus is more central than CIS;    -   5. nucleus may follow shape of cytoplasm; nucleus shows less        distortion than CIS;    -   6. nuclear pleomorphism is increased with coarse chromatin        present and condensation along nuclear envelope;    -   7. parachromatin, large nucleus, multi-nuclustered nuclear        membrane focally thickened; and    -   8. cells show predominant acidophilic cytoplasm.

Carcinoma in-situ squamous (CIS, non-invasive). Cells were identified asbeing carcinoma in situ squamous if the following criteria weresatisfied:

-   -   1. cells single or in aggregates (clumps);    -   2. cell size variable—may be smaller or larger than marked        dysplasia cells usually smaller than invasive squamous cell        carcinoma;    -   3. cells are large, rounded with symmetrically located nucleus;    -   4. cell degeneration may be present;    -   5. scant cytoplasm, distributed uniformly maybe keratinized or        non-keratinized concentrically around the nucleus,        (orangiophilic or basophilic);    -   6. N/C ratio variable—higher or lower than normal;    -   7. coarse dense nuclear chromatin granules may be interrupted by        clear zones;    -   8. uniformly thickened chromatinic rim with undulation of        nuclear membrane;    -   9. lobulations of nuclei may be seen;    -   10. cannibalism may be seen, but is unusual;    -   11. multinucleated cells may be present;    -   12. no nucleoli in nucleus; a mitotic cell may be present; and    -   13. clean background.

Squamous cell carcinoma (well differentiated keratinizingtype-invasive). Cells were identified as squamous cell carcinoma if thefollowing criteria were satisfied:

-   -   1. cells usually single, orangeophilic, but may be in clusters,        and degenerate;    -   2. cells large or small, angular, with well preserved nuclei and        distinct cell borders;    -   3. cells usually larger than in-situ, and may be pleomorphic,        wide range of both size and shape;    -   4. pearl formation may be seen (cancer pearl);    -   5. moderate amount of cytoplasm with abnormal “tailing”        (consistent with invasion); bizarre cell shapes—tadpole, star,        spindle; a nuclear chromatin angular, with unpredictable        clumping with hyperchromasia and parachromatin clearing and        clearly defined chromatin, parachromatin interface;    -   6. chromatin is coarsely clumped, especially along the nuclear        membrane;    -   7. nucleoli are large and acidophilic, if present;    -   8. nuclear membrane itself may be thickened and irregular;        irregularity of thickness of nuclear chromatin rim;    -   9. N/C ratio is very high;    -   10. marked nucleolar irregularity in shape, size, numbers        (daughter nucleoli); abnormal mitoses, multi nucleation;    -   11. cannibalism and multinucleation is common; and    -   12. necrotic background material is common.        Results

In a blinded study in which 60 samples were examined, the resultsindicate that abnormal cells (mild, moderate or severe dysplasia orcancerous) can be accurately detected with the TCPP detection procedurecompared with the PAP-staining procedure (Table 1). If 2-3% of theTCPP-exposed cells were fluorescing, then the sample reliably correlatedwith at least the mildly dysplastic diagnosis. Fifty out of fifty sputumsamples determined by the standard cytomorphological PAP-stainingprocedure to be mildly dysplastic to cancerous were also identified asabnormal by TCPP detection. Among the ten samples characterized asnormal or metaplastic based on the PAP-staining procedure, four samplesdemonstrated that same morphology using the TCPP method. Samplesdiagnosed as normal showed minimal or no TCPP uptake.

TCPP uptake in cells determined to be negative or metaplastic bycytomorphology had characteristic fluorescence intensity and patternsthat were recognizable and diagnostic. Table 2 presents a comparisonbetween cell morphology and fluorescence as determined by PAP-stainingcytomorphology and TCPP techniques, respectively. Based on fluorescenceintensity and pattern in TCPP-treated cell samples, cells werecategorized with one of 14 possible numbered-classifications relating toa morphological description. If cells were class 11 using the TCPPdetermination and fewer than 2-3% of the cells on the slide werefluorescent above background levels, then that sample was determined tobe metaplastic and not dysplastic. The metaplastic cells were easilydifferentiated from normal cells by their moderate fluorescence with abarely visible cell membrane. Of the ten cell samples that weredetermined to be negative or metaplastic by PAP-staining, 6 weredesignated with a class 11 cell description based on TCPP fluorescence.Of the six TCPP samples with class 11 designation, three also indicatedfluorescence from nuclear debris (i.e., either a class 13 or 14), andtwo also showed class 10 designation (fluorescence from the nucleusonly).

Another pattern shown in Table 2 relates to numerical classification6—Irregular anisoid cells, low to medium fluorescence. It is notablethat relatively few dysplastic cells were assigned this classification,while most carcinomic cells received classification 6. Hence, thisclassification is expected to be of particular importance indistinguishing carcinomas from dysplasias using the methods of theinvention.

Another significant observation revealed in Table 2 is that, as cellmorphology progressed from normal to severely carcinomic, the totalnumber of numerical classifications that were assignable to eachexamined cell also increased. As an illustration, cells having anegative or metaplastic morphology were assigned an average of 2numerical classifications, while cells displaying adenicarcinoma,squamous cell and small cell carcinoma were assigned an average of 5numerical classifications. Since the numerical classifications containdescriptions of different kinds of cellular abnormalities, a positivecorrelation between degree of dysplasia or carcinoma and the number ofdifferent abnormalities observed in the cells is logical. However, sucha correlation heretofore has not been systematized and used to diagnoseprecancerous and cancerous conditions in a sample of cells.

TABLE 1 Correlation Between TCPP Results and Cytomorphological Results.N = 60 Slides with morphology using TCPP/ Diagnosis Description Slideswith morphology using cytomorphology Negative or metaplastic  4/10 Milddysplasia 12/12 Moderate dysplasia 9/9 Severe dysplasia 8/8 Carcinoma insitu 11/11 Adenocarcinoma, 10/10 squamous cell and small cell carcinoma

TABLE 2 Cell descriptions: Cytomorphological Characteristics and TCPPFluorescence. Number of Samples with Cells having Numerical Descriptionby TCPP Fluorescence Microscopy/Number of Samples with Cell Descriptionby Cytmorphology Adenicarcinoma, squamous cell Negative or Mild ModerateSevere Carcinoma and small cell metaplastic dysplasia dysplasiadysplasia in situ carcinoma Classification Number = Cell description (n= 10) (n = 12) (n = 9) (n = 8) (n = 11) (n = 10)  1 = Large nucleus ornuclei, low to medium fluorescence 3/10 12/12  9/9 7/8 10/11  6/10  2 =Symmetrical binuclear cells, medium fluorescence 0/10 5/12 5/9 3/8 3/112/10  3 = Small oval cells, medium to high fluorescence 0/10 4/12 4/92/8 6/11 6/10  4 = Small round cells, low fluorescence 0/10 0/12 0/9 0/80/11 2/10  5 = Multi-nucleated cells, medium fluorescence 0/10 0/12 3/96/8 9/11 7/10  6 = Irregular anisoid cells, low to medium fluorescence0/10 0/12 1/9 1/8 10/11  7/10  7 = Cellular clusters, medium to highfluorescence 0/10 0/12 1/9 0/8 1/11 3/10  8 = Degenerated single cells,medium to high fluorescence 0/10 0/12 0/9 0/8 4/11 7/10  9 = Degeneratedcell clusters, medium to high fluorescence 0/10 0/12 0/9 0/8 1/11 4/1010 = Cells uniform size with small round nucleus, medium 2/10 2/12 2/91/8 1/11 2/10 fluorescence (nucleus only) 11 = Cell membrane barelyvisible, medium fluorescence 6/10 6/12 2/9 2/8 2/11 0/10 12 = Nucleardebris clumps, no fluorescence 0/10 1/12 0/9 0/8 0/11 0/10 13 = Nucleardebris background, no fluorescence 6/10 5/12 8/9 2/8 5/11 3/10 14 =Nuclear debris background, medium fluorescence 1/10 2/12 2/9 1/8 1/110/10 Avg. # numerical descriptions per cell examined 1.80 3.08 4.11 3.134.82 4.90 Each cell sample may be designated with more than one of the14 numerical cell descriptions for TCPP-treated cells.Suspension Assay for Detection and Separation of Pre-Cancerous andCancerous Cells Using TCPP

This example discloses the use of TCPP staining in conjunction withfluorescence flow cytometry in combination with cytomorphologic slidemicroscopy to determine the abnormality of cells found in sputumsamples. By virtue of the specificity of TCPP staining, the combinationof flow cytometry followed by slide microscopy is particularly powerful,providing an internal control for cytomorphologic slide comparisons.

Methods

Sputum Processing Procedures Used in Production of Suspensions andMonolayer Slides. All suspensions and monolayer slides are produced fromsputum samples collected through patients performing the early morning,spontaneous cough technique. Specifically, patients are instructed toexpectorate whatever material they cough up across three consecutivemornings into a container filled with fixative consisting of 2% Carbowaxin 50% alcohol/50% Saccomanno fluid with 0.03-0.05 mg/mL rifampin.Rifampin is added to the fixative solution to serve as a prophylacticagainst patients harboring M. tuberculosis or those patients who may beasymptomatic carriers of N. meningitis.

The 2% Carbowax solution is prepared by adding 2 mL melted Carbowax(150) to 98 mL of 50% ethanol and mixing for 30 minutes. Glassware usedto make solution is kept warm to prevent hardening of wax on the surfaceduring preparation, which can cause inaccurate measurement. Carbowax isremoved before exposure to the TCPP working solution by immersion in 95%alcohol for at least 15 minutes.

Rifampin solution (3 mg/mL) is made by dissolving 300 mg capsules ofrifampin into 100 mL ethyl alcohol and blending at high speed in aWaring blender. One mL of this solution is added to each 30 mL ofSaccomanno solution or 20 mL per liter of Saccomanno solution and mixedthoroughly. The preparation of Saccomanno solution is according tostandard methods well known to those in the art of cytology.

The sputum specimen is poured into a 50 mL plastic centrifuge tube andadditional 50% ethyl alcohol solution added to bring the volume to 50 mLif necessary. The contents of the centrifuge tube are poured into a 250mL Eberbach semi-microblender container and homogenized for 10 to 60seconds, depending upon visual examination of the specimen and mucoidcontent. Thick mucoid specimens sometimes required longer blendingtimes. The specimen is poured back into the centrifuge tube andcentrifuged at low speed for 10 minutes. The supernatant is decanted,leaving 1 to 2 mL in the centrifuge tube to admix with the cell pellet.The tube is agitated on a vortex mixer for approximately 10 seconds. Thesample is resuspended in 100 mM MES buffer, pH ˜6.15. The cells are spunand rinsed two more times with 100 mM MES buffer, the last time leaving˜1 mL in the centrifuge tube in which to resuspend the cells. The cellsare then resuspended in 15 ml 95% ethanol (5% 100 mM MES buffer) at roomtemperature (˜20° C.) for 30 minutes, with gentle agitation. Thecentrifuge tube is then centrifuged at low speed for 10 minutes. All but1-2 ml of the supernatant is removed.

TCPP Stock Solution. 400 mg of sodium bicarbonate is added toapproximately 90 mL of the 50% isopropanol (50 mM sodium bicarbonate)and mixed until completely dissolved to make basified 50% isopropanol.One hundred milligrams of TCPP are slowly add to the basified 50%isopropanol (50 mM sodium bicarbonate) and mixed for 3 to 5 minutesuntil dissolved. The TCPP solution is brought to 100 mL volumetricallywith the basified 50% isopropanol, mixed well and stored in an amberreagent bottle covered in foil in a refrigerated area. The finalconcentration of TCPP in the stock solution is 1 mg/mL.

TCPP Working Solution. Fresh TCCP working solution is prepared each day.Approximately 10 mL of TCPP Stock Solution with concentration 1 mg/mL isbrought to room temperature. Eight milliliters of the TCPP StockSolution (1 mg/mL) are placed in a 200 mL volumetric flask andapproximately 100 mL of the MS buffer is slowly added. The solution isgently mixed. Additional MES buffer is added to bring the solution to200 mL volumetrically. The solution is mixed for 3 to 5 minutes andstored at 2-4° C. in an amber bottle. The final concentration of TCPP inthe working solution is 40 μg/mL.

TCPP Exposure Procedure. Suspension cells, previously exposed to 95%alcohol for 30 minutes at room temperature, are exposed to TCPPimmediately after fixing or up to 3 days later. The cells areresuspended in 10 ml of 40 μg/mL TCPP solution for 10 minutes at 36° C.with gentle agitation, then washed with 20 ml of room temperature, 100mM MES buffer three times, using centrifugation at minimum speed toloosely pellet the cells within 10 minutes. The washed cell pellet isresuspended in 15-10 ml of MES buffer. These suspensions, or aliquotsthereof, are passed through a fluorescence flow cytometry apparatus.

Fluorescence flow cytometry. First Pass. A minimum of 10,000 cells arepassed through a flow cytometer with cell sorting capability. The flowcytometer should be equipped with a light source providing radiation atabout 415 nm, with filters to allow passage of light between about 390nm and 490 nm. Fluorescence emission should be monitored between about630 nm and 730 nm (emission maxima at 645 nm and 706 nm). A barrierfilter passing light above 500 nm is satisfactory. On the first pass,individual cells are counted and their specific fluorescence measured.The average fluorescence is calculated, and the standard deviation fromthat average is calculated. Also, the median value is determined (thespecific fluorescence value that is smaller than half the values andgreater than half the values).

Fluorescence flow cytometry with cell sorting. Second Pass. A minimum of100,000 cells are passed through the fluorescence flow cytometer withcell sorting capability, equipped the same as for the first pass. Cellswith fluorescence less than the Median fluorescence +1.3 standarddeviations from the mean (approximately 90% of the cells) areoperationally defined as having low fluorescence, and are saved in onetest tube, and cells with specific fluorescence greater than or equal tothe median fluorescence +1.3 standard deviations from the mean(approximately 10% of the cells) are operationally defined as havinghigh fluorescence and are saved in another test tube. Alternatively,cells can be sorted into tubes according to their fluorescence relativeto the median specific fluorescence found in the first pass. Cells withless than twice the median specific fluorescence would be “normal” orlow fluorescence, and then cells could be pooled with 2-4×, the medianfluorescence, 4-6×, and greater than 6× median fluorescence. Each of thehigher intensity fluorescence pools would be expected to be more greatlyenriched in abnormal cells. If more than 2-3% of the cells possessedmore than 3× the median fluorescence, there would be support for apresumption of at least an advanced precancerous condition.

Production of Monolayer Slides. The low- and high-fluorescent cellsamples are centrifuged for 10 minutes at low rpm to pellet the cells.The supernatant is removed, leaving 1-2 mL in the centrifuge tube toadmix with the cell pellet. The tube is agitated on a vortex mixer forapproximately 10 seconds. One to three drops of the sediment is placedinto a PreservCyt vial (Cytyc Corporation, Marlborough, Mass.). Thespecimen is incubated for 5 minutes to deactivate all microbial andviral organisms.

Monocellular layers of the samples are fixed onto slides using theThinprep Processor (CYtyc Corporation, Marlborough, Mass.) according tothe manufacturer's instructions. In the ThinPrep Processor, cells arecollected onto a polycarbonate filter (pore size 0.5 mm) and transferredto a glass slide. The ThinPrep Processor then immediately deposits theslides into a fixative bath containing 95% ethanol (maintain for 30minutes).

Sputum Slide Staining Procedures by Modified PAP-Staining Technique.Procedure sequence (no.), reagent and time (min.:sec.) are as follows:(1) 95% alcohol 15:00; (2) tap water 1:00; (3) gil-i hemotox 2:30; (4)tap water 1:00; (5) bluing reagent: 30; (6) tap water 1:00; (7) 95%alcohol: 10; (8) og-6 1:30; (9) 95% alcohol: 10; (10) 95% alcohol: 10;(11) ea-50 1:15; (12) 95% alcohol: 20; (13) 95% alcohol: 30; (14) 100%alcohol 1:00; (15) 100% alcohol 1:00; (16) 100% alcohol 1:30; (17)xylene 1:00; (18) xylene 1:00; and (19) xylene 1:00.

Methods for Routine Cytopathological Analysis of Papanicolaou-StainedSlides. PAP-stained slides undergo semi-quantitative cytomorphologicalevaluation. (1) dysplastic and neoplastic cells are identified throughuse of traditional morphologic criteria, and (2) the expression levelsof seven fundamental indicators of pulmonary inflammation (alveolarmacrophages, neutrophils, columnar cells, mucus, mucous spirals,pigmented macrophages, metaplastic cells) are quantified. Themethodology for quantifying these inflammation indicators have beenpreviously discussed in the literature (Roby et al., 1989, Acta Cytol34:147-154; Roby et al., 1990, Acta Cytol 34:140-146; Schumann et al.,1989, Am Rev Respr Dis 139:601-603). The criteria used to determine cellmorphology using PAP-stained cytology are discussed below.

No significant abnormalities. Cells are identified as having nosignificant abnormalities if the following criteria are satisfied:

-   -   1. basophilic, ciliated epithelial cells admixed with        macrophages with grade 1-2 pigment along with inflammatory        cells;    -   2. round nuclei of epithelium basally oriented;    -   3. evenly dispersed chromatin;    -   4. inconspicuous nuclear membranes;    -   5. inconspicuous nucleoli; and    -   6. no metaplastic and no dysplastic cells present.

Squamous metaplasia (without dysplasia). Cells are identified assquamous metaplastic without dysplasia if the following criteria aresatisfied:

-   -   1. clumps of basophilic cells without cilia;    -   2. uniform cell and nuclear size;    -   3. low nucleus/cytoplasm (N/C) ratio;    -   4. nuclear chromatin finely granular; and    -   5. small rounded nucleoli (usually single) may be present.

Mild dysplasia (squamous atypia). Cells are identified as mildlydysplastic if the following criteria are satisfied:

-   -   1. smaller than metaplastic cells;    -   2. seen in cohesive clusters, or singularly;    -   3. cells lie flat (sheets) both nuclei and cytoplasm in focus;    -   4. cells vary slightly in size;    -   5. cytoplasm may be eosinophilic or basophilic;    -   6. cytoplasmic borders sharp;    -   7. nuclei vary slightly in size, usually round to oval, if        divided 2 halves of nucleus are mirror images, N/C ratio may        vary slightly;    -   8. nuclear membrane smooth;    -   9. nuclear chromatin (slightly increased) finely granular,        occasional chromocenter; and    -   10. fiber cells, elongated cells with stretched cytoplasm and        nucleus distinct nuclear membrane—fine reticular to granular        cytoplasm usually bright yellow orange—keratinizing single, may        form whorls around central core of keratin to make epithelial        pearls.

Moderate dysplasia (squamous atypia). Cells are identified as moderatelydysplastic if the following criteria are satisfied:

-   -   1. variation in size, usually larger but may be smaller than        mild dysplasia;    -   2. more variation in shape and N/C ratio than mild dysplasia;    -   3. cytoplasm dense, acidophilia predominates; increased number        of atypical cells;    -   4. nucleus may have unequal halves (not mirror images);    -   5. nuclear lobulations, crevices, and nodules are present; and    -   6. nuclear material may show hyperchromasia with more        stippled—like chromatin pattern.

Severe dysplasia (marked squamous atypia). Cells are identified asseverely dysplastic if the following criteria are satisfied:

-   -   1. cells vary markedly in size and shape;    -   2. usually slightly larger cell size than moderate dysplasia;    -   3. N/C ratio is high but variable (with extremes);    -   4. single cells predominate; nucleus is more central than CIS;    -   5. nucleus may follow shape of cytoplasm; nucleus shows less        distortion than CIS;    -   6. nuclear pleomorphism is increased with coarse chromatin        present and condensation along nuclear envelope;    -   7. parachromatin, large nucleus, multi-nuclustered nuclear        membrane focally thickened; and    -   8. cells show predominant acidophilic cytoplasm.

Carcinoma in-situ squamous (CIS, non-invasive). Cells are identified asbeing carcinoma in situ squamous if the following criteria aresatisfied:

-   -   1. cells single or in aggregates (clumps);    -   2. cell size variable may be smaller or larger than marked        dysplasia cells usually smaller than invasive squamous cell        carcinoma;    -   3. cells are large, rounded with symmetrically located nucleus;    -   4. cell degeneration may be present;    -   5. scant cytoplasm, distributed uniformly maybe keratinized or        non-keratinized concentrically around the nucleus,        (orangiophilic or basophilic);    -   6. N/C ratio variable—higher or lower than normal;    -   7. coarse dense nuclear chromatin granules may be interrupted by        clear zones;    -   8. uniformly thickened chromatinic rim with undulation of        nuclear membrane;    -   9. lobulations of nuclei may be seen;    -   10. cannibalism may be seen, but is unusual;    -   11. multinucleated cells may be present;    -   12. no nucleoli in nucleus; a mitotic cell may be present; and    -   13. clean background.

Squamous cell carcinoma (well differentiated keratinizingtype-invasive). Cells are identified as squamous cell carcinoma if thefollowing criteria are satisfied:

-   -   1. cells usually single, orangeophilic, but may be in clusters,        and degenerate;    -   2. cells large or small, angular, with well preserved nuclei and        distinct cell borders;    -   3. cells usually larger than in-situ, and may be pleomorphic,        wide range of both size and shape;    -   4. pearl formation may be seen (cancer pearl);    -   5. moderate amount of cytoplasm with abnormal “tailing”        (consistent with invasion); bizarre cell shapes—tadpole, star,        spindle; a nuclear chromatin angular, with unpredictable        clumping with hyperchromasia and parachromatin clearing and        clearly defined chromatin, parachromatin interface;    -   6. chromatin is coarsely clumped, especially along the nuclear        membrane;    -   7. nucleoli are large and acidophilic, if present;    -   8. nuclear membrane itself may be thickened and irregular;        irregularity of thickness of nuclear chromatin rim;    -   9. N/C ratio is very high;    -   10. marked nucleolar irregularity in shape, size, numbers        (daughter nucleoli); abnormal mitoses, multi nucleation;    -   11. cannibalism and multinucleation is common; and    -   12. necrotic background material is common.

Cytopathology Analysis. Because sputum samples contain cells from manylocations in the lung, interspersed with each other, there is littlecontext for judging the normality or abnormality of a particular cell(unlike the case for thin section staining). The availability of acollection of low-fluorescence cells provides an internal control sampleof normal or nearly normal patient cells, with which to compare thehigh-fluorescence TCPP-stained cells. Using the standard PAP-stain, acytopathologist skilled in the art can readily determine the degree ofabnormality of the high-fluorescence TCPP cells, which are 10-foldenriched for abnormal cells compared with an un-fractionated monolayer.

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

1. A method of prognosing a patient's response to a cancer therapy, themethod comprising: a) prior to the therapy, contacting a sample of cellsfrom the patient's tissue or organ being treated for the cancer with asolution of 5,10,15,20-Tetrakis (Carboxyphenyl) Porphine (TCPP) underconditions permitting binding of the TCPP to components of abnormalprecancerous or cancerous cells, if any are present; b) detecting TCPPfluorescence in the sample, the presence of TCPP fluorescence beingindicative that the sample contains dysplastic or carcinomic cells; c)at intervals during the therapy and subsequent to the therapy,performing steps a) to b) on another sample of cells from the patient'stissue or organ being treated for the cancer; and d) determining if thepercentage of the abnormal pre-cancerous or cancerous cells in thesamples tested during and subsequent to the therapy are reduced ascompared with the sample tested prior to the therapy, the reductionbeing prognostic of the patient's response to the cancer therapy.
 2. Themethod of claim 1, further comprising separating the cells byfluorimetric flow cytometry into a population comprising normal cellsand a population comprising abnormal precancerous and cancerous cells.3. A method of prognosing a patient's response to a cancer therapy, themethod comprising: a) prior to the therapy, contacting a sample of cellsfrom the patient's tissue or organ being treated for the cancer with asolution of 5,10,15,20-tetrakis (carboxyphenyl) porphine (TCPP) at aboutroom temperature for between about 0.2-3.0 minutes to permit binding ofthe TCPP to components of abnormal precancerous or cancerous cells, ifany are present, wherein the solution of TCPP comprises the TCPPpredissolved in basified alcohol; b) detecting TCPP fluorescence in thesample, the presence of TCPP fluorescence being indicative that thesample contains abnormal precancerous and cancerous cells; c) atintervals during the therapy and subsequent to the therapy, performingsteps a) to b) on another sample of cells from the patient's tissue ororgan being treated for the cancer; and d) determining if the percentageof the abnormal pre-cancerous or cancerous cells in the samples testedduring and subsequent to the therapy are reduced as compared with thesample tested prior to the therapy, the reduction being prognostic ofthe patient's response to the cancer therapy.
 4. The method of claim 3,further comprising separating the cells by fluorimetric flow cytometryinto a population comprising normal cells and a population comprisingabnormal precancerous and cancerous cells.